410
Anal. Chem. 1983, 55, 410-411
Isolation of Aquatic Humus with Diethylaminoethylcellulose Carl J. Miles," John R. Tuschall, Jr.,' and Patrick L. Brezonik2 Department of Environmental Engineering Sciences, University of Florida, Gainesville, Florida 326 1 1
The chemical nature of aquatic humus has been the subject of much research. Because of the dilute nature of natural water-humus solutions, interest has centered on methods to isolate and concentrate these substances for further studies (1-3). Several methods of isolating or concentrating humic substances are available, including freeze concentration ( 4 , 5 ) , chemical precipitation (6, 7), solvent extraction (8,9) and adsorption t o solids ( I , 10-13). Isolation and concentration of humics by adsorption to solids (Le., XAD, ion exchange resins) are desirable because of the selectivity of available adsorbents. Macroreticular resins such as XAD have been studied and used extensively (1, 3, 14), but their use requires lengthy resin preparation. Humic material has a weak acidic character, and this property can be used by employing anion exchange materials. Plastic bead-type ion exchange resins (i.e., poly(styrene-divinylbenzene)) are readily available, but reports of irreversible binding, leaching of native organics, and destruction of biopolymers such as DNA have made these media unattractive. Ion-exchange celluloses, on the other hand, are easy to pretreat and have desirable morphological properties. Furthermore, sorption of inorganic ions is minimal a t pH -7 ( 4 ) . Our laboratory has been investigating aquatic humus in surface waters with a range of concentration up to about 100 mg L-l. For various experiments we have the need to isolate and prepare large quantities of these materials. Currently, we are using the weak anion exchange material, diethylaminoethylcellulose (DEAE-cellulose), to isolate and concentrate humic substances from natural waters. This material has been proposed for use in one step of a fractionation scheme for natural organic matter ( 4 ) , but its characteristics and limitations in isolation of humics have not been reported to date. Our intent is to describe a procedure to isolate humics from water and to report the efficiency and useful pH range of DEAE-cellulose in both column and batch modes.
EXPERIMENTAL SECTION Natural waters used in this study were collected from Hatchett Creek, Hogtown Creek, Santa Fe River, and Suwannee River in northcentral Florida. Chemical characteristics of these waters are summarized in Table I. All chemicals were analytical reagent grade or better. Glassware was silanized by soaking in a 5% solution of dichlorodimethylsilanein CCll overnight. Afterward, the glassware was rinsed with methanol. All pH adjustments were made with 1M solutions of NaOH and HC1, and pH was measured with a Model 91-15 Orion electrode connected to a Model 401 Orion pH meter, Dissolved organic carbon (DOC) measurements were made on fiitered and acidified samples with an Oceanography International (01)Corp. Model 524B-HR direct inject module attached to an 01 524C totalorganic carbon analyzer. Ultraviolet absorbance was measured at pH 7.0 on a Perkin-Elmer Model 552 UV/VIS spectrophotometer using a 1.0-cm SPECTROSIL cuvette. DEAE-cellulose (Eastman) was pretreated by the following procedure. About 50 g of dry cellulose was mixed in 1000 mL of 0.5 M HCl for 1h. The cellulose was rinsed with deionized water (DIW) in a Buchner funnel until the pH was neutral. The cellulose was resuspended in 0.5 M NaOH for 1h and rinsed with DIW until the pH was neutral. Fines were removed by suspending small amounts (ca. 5-10 g dry weight) in a 1000-mL graduated cylinder of DIW and settling 1.0 h before removing the super'Present address: Illinois State Water Survey, P.O. Box 5050, Station A, Champaign, IL 61820. Present address: Department of Civil and Mineral Engineering, University of Minnesota, Minneapolis, MN 55455.
Table I. Chemical Characteristics of Natural Waters Used in This Study and Percent Recovery of Aquatic Humus from Each % recovery by
color
a
n W A W
site
pH
units)
Hatchett Creek Hogtown Creek Santa Fe River Suwannee River
6.1
180
19.3
89
89
7.8
53
15.8
87
91
7.4
303
46.0
86
85
7.3
333
49.0
88
86
L"
column batch
Based on DOC.
natant solution. The pretreated cellulose was stored in darkness at 4 "C. Humic materials from Hatchett Creek that were isolated by the DEAE method were desalted on Chelex 100 (H' form) and freeze-dried prior to the following chemical characterization. An infrared spectrum was recorded on KBr pellets with a PerkinElmer Model 337 IR spectrometer and elemental analysis was made with a Perkin-Elmer Model 240 CHN analyzer. Total and carboxyl acidity tritrations were performed by the methods described by Schnitzer and Khan (15)and average molecular weights were determined by gel filtration chromatography by using Sephadex G-15 with 0.1 M K2HP04buffer (pH 9.0). Gas chromatography/mass spectrometry (GC/MS) analysiswas performed on methylated (CH2N2)humic materials with a Hewlett-Packard Model 5985B GC/MS equipped with a fused silica capillary column. High-performance liquid chromatography was done on a Perkin-Elmer Series 2 LC with a 5 fim ODS stationary phase and 5% acetic acid solution as the mobile phase. Column Procedure. Pretretated DEAE-cellulose (0.3 g dry wt) was placed in a beaker containing DIW and adjusted to the desired pH. The slurry was poured into a 1.1X 8 cm glass column with reservoir. The column was rinsed with DIW at the pH of interest until pHh = pHout,and the effluent DOC was less than 2 mg L-l. A 1.0-L aliquot of humic water (adjusted to the pH of interest) was applied to the column at about 2 mL m i d . After the column was rinsed with 2 bed volumes of DIW, the retained material was desorbed with 20 mL of 0.1 M NaOH and neutralized with concentrated HC1. Samples were analyzed for DOC and UV absorbance to determine recovery. In addition, when it was necessary to desalt the isolated humic compounds prior to further investigation,elution through a Chelex 100 column (H' form) was found to effectively reduce the salt concentration with excellent (>95%) recovery of humic material. Batch Procedure. Pretreated DEAE-cellulose (0.3 to 0.6 g dry wt) was placed in a beaker, and the pH was adjusted t o 6.0. A 1.0-Laliquot of humic water (at pH 6) was added t o the beaker and stirred for 30 min. Settling was complete within 1h. After the supernatant was decanted, the slurry was packed into a 1.1 X 8 cm column rinsed with 2 bed volumes of DIW, desorbed with 20 mL of 0.1 M NaOH, and the pH of the eluant was lowered to 0
1.
RESULTS AND DISCUSSION We use the term aquatic humus to describe the complex polycarboxylate-polyphenolic acids found in natural waters that are similar to terrestrial humic substances (i.e,, fulvic acid). Humic molecules are dynamic; they are synthesized from precursor molecules as well as degraded to smaller subunits. Aquatic humus has traditionally been associated
@ 1983 American Chemlcai Society 0003-2700/83/0355-0410$01.50/0
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983 I00
8C
>
60
w
$ LL
8
4c
20
2
1
6
8
10
PH
Figure 1. Recovery of color and DOC from column and batch treated Hatchett Creek water as a function of pH.
with organic color but some uncolored low molecular weight complex acids have been found in natural waters (16). It seems likely that the DEAE procedure described here isolates all natural complex acids whereas the XAD technique does not concentrate the colorless ]lowmolecular weight specific organic acids in water. The humic material that we isolated from Hatchett Creek by the DEAE techniques had the following chemical-physical characteristics; (1)Spectroscopic data. Major infrared bands were 3400 cm-' (OH), 1700 cm-.' (C=O of COOH; C=O ketonic), 1580 cm-l (aromatic C=C, H bond C=O), and 1375 cm-I (COO-Na+). Visible spectra showed the ratio of absorbance a t 465 nm to 665 rim ( E 4 / & ratio) to be 8.8. Aquatic humus is considered to be similar to soil fulvic acid and the E,/& ratio of the latter is about 10 (15). The E,/& values for the other three whole water samples ranged from 9.6 to 10.5. (2) Elemental ana1,ysis. Values reported are corrected for the percentage of ash: carbon, 48.4%; hydrogen, 5.67%; nitrogen, 1.96%. (3) Acidity titrations. Titrations yielded a value of 13.6 mequiv/g for total acidity and 10.0 mequiv/g for carboxyl acidity. (4) Molecular weight. Based on gel filtration chromatography the average molecular weight range of the form water samples was lo3 to 1.5 X IO3daltons. Other aquatic humus samples that we isolated by this method had similar chemical-physical characteristics. Furthermore, GC/MS and HPLC analyses of the Hatchett Creek water did not reveal any nonhumified organic acids (Le., steric, palmitic) above the microgram per liter level. The humic molecule is considered to derive most of its acidic character from carboxyl and hydroxyl functional groups (15).These weak acids are well retained by the DEAE-cellulose at neutral pH values. Figure 1shows that the optimum recovery of Hatchett Creek organic acids occurred in the pH 4-6 range. Both DOC and UV absorbance (300 nm were used to monitor recovery, and tlhe two parameters gave comparable results. Efficiency dropped off at low pH values because the protonated organic acids were no longer anionic and thus were not retained by DEAE-cellulose. At high pH, efficiency also decreased because of neutralization of the amino moiety on DEAE-cellulose. For the intermediate pH levels, the absorbed humics created a dark brown band at the head of the column, which contrasted sharply with the white, lower portion of the column. However, at extreme pHs (2 and lo), the brown band was less distinct due to mibTation of some humics through the
411
column. These results are expected based on purely ionic interactions between the solute and exchange material. Unlike the XAD resins, the mechanism of retention of humics to DEAE-cellulose appears to be based on charge rather than hydrophobic interactions. However, it is also possible that the mechanisms of sorption of organic anions by DEAEcellulose is by hydrogen bonding through the phenyl group on humic material (17). Table I lists the percent recovery of aquatic humus at pH 6 from the four sources of natural waters selected. All recoveries were better than 85% based on DOC; recoveries based on UV absorbance gave comparable results. A method blank had no detectable UV absorbance and only 0.8 mg L-' of DOC. This level of contamination was less than 6% of the total DOC desorbed for the worst-case sample water (i.e., Hogtown Creek). For waters with higher humic concentrations, this amount of bleed represents a much lower percentage of total DOC desorbed. Table I shows that recovery of aquatic humus by the batch method was comparable to the column method and in one case slightly higher. Since one would not expect the column and batch recoveries to be similar under the same experimental conditions, it is possible that a small fraction of aquatic humus is unrecoverable from either procedure. The batch method is simpler to perform than the column method, and in cases where recoveries are similar, the batch method can be easily used to concentrate humics in the field. In summary, the anion exchange material, DEAE-cellulose, has been shown to rapidly and efficiently concentrate and isolate organic acids from natural waters at pH levels from 4 to 8. Inorganic anions, such as chloride and bicarbonate, a t levels typically found in freshwater, are not concentrated by this method and thus DEAE-cellulose serves to both concentrate and separate organic acids from most other anions, cations, and neutral species in natural waters. In addition, because of its simplicity, the batch technique is particularly suitable for concentrating humic compounds in the field.
ACKNOWLEDGMENT The authors wish to thank Vickey Maxfield for preparation of this manuscript. Registry No. DEAE, 9013-34-7;water, 7732-18-5. LITERATURE CITED (1) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Techno/. 1981, 15, 463-466. (2) Leenheer, J. A. Environ. Sci. Technol. 1981, 15,578-587. (3) Aiken, G. R.; Thurman, E. M.; Malcolm, R. L.; Walton, H. F. Anal. Chem. 1979, 51, 1799-1803. (4) Slrotklna, I. S.;Varshall, G. M.; Lure, Y. Y.; Stepanova, N. P. Zh. Anal. Khim. 1974, 29, 1626-1632. (5) Shaplro, J. Science 1981, 133, 2063-2064. (6) Jeffery, L. N.; Hood, D. W. J . Mar. Res. 1958, 17,247-269. (7) Martin, F. D.; Pierce, R. H. Envlron. Letf. 1971, I , 49-58. (8) "Drinking Water and Health"; Natlonal Academy of Sciences: Washington, DC, 1977; pp 489-856. (9) Baker, R. A.; Malo, B. A. J . Sanit. Eng. Div., Am. SOC. Civ. Eng. 1967, 93,41-54. (IO) Levesque, M.; Schnitzer, M. Soil Sci. 1987, 103, 183-190. (11) Ogner, G.; Schnltzer, M. Can. J . Chem. 1971, 49, 1053-1063. (12) Christman, R. F.; Chassemi, M. J.-Am. Water Works Assoc. 1988, 58,723-741. (13) Rebhun, M.; Manka, J. Environ. Sci. Technol. 1971, 5,606-609. (14) Manotura, R. F. C.; Riley, J. P. Anal. Chim. Acta 1975, 7 6 , 97-106. (15) Schnltzer, M.; Khan, S. U. "Humic Substances in the Environment"; Marcel Dekker: New York, 1972. (16) Malcolm, R., personal communication, 1982. (17) Kim, B. R.; Snoeylnk, V. L.; Saunders, F. M. J.-Water Pollut. Control Fed. 1978. 4 8 , 120-133.
RECEIVED for review June 30, 1982. Resubmitted November 1, 1982. Accepted November 12, 1982.
